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Abstract To provide reference for selenium resource exploitation of soil， many selenium-tolerant strains were isolated from selenium-rich soil in Guangxi. The dilution spread plate method and selenium-added culture method were used to screen the selenium-tolerant strains from the soils which were sampled from the main Selenium-rich areas such as Yongfu， Bama， Yulin Hanshan， Guiping and Tengxian. The results showed that 8 strains with high selenium tolerance were obtained， which could tolerate the selenium concentration above 10 000 μg/m L in solid medium. Among the 8strains， YLB1-33 showed the highest selenium tolerance， and it could still grow weakly in the solid medium with selenium concentration of 29 000 μg/m L. The sequencing of 16S rDNA and phylogenetic analysis showed that YLB1-6 was identified as Bacillus cereus， BMB2-1 and TXB1-8 were identified as Bacillus pumilus， GPB2-5 was identified as Bacillus thuringiensis， YLB1-26 and YLB1-33 were identified as Bacillus licheniformis， and YLB1-2 and YFB1-8 were identified as Serratia marcescens. The finding of selenium-tolerance strains had potential application value on promoting the utilization of selenium soil resources and the development of selenium-rich agricultural products in Guangxi.
Key words Selenium-rich soil； Selenium tolerance； Strains； Identification； Guangxi
Selenium （Se） is one of the 15 trace elements essential to the human body. It is particularly important to meet the demand of peoples daily supplement of selenium by properly exploiting and utilizing the selenium resources in soil to produce natural selenium-enriched agricultural products. Guangxi is a large selenium-riched province with an area of 2.12 million hm2 of selenium-riched soil. It is the area of large contiguous selenium-riched soil currently delineated in China[1]， where the soil selenium content can reach 2.29 mg/kg at the most [2]. However， because selenium-riched soil is more widely distributed in moderately acidic soils in Guangxi， selenium is easy to form oxides and hydrous oxides with extremely low solubility with iron， which greatly reduces the available selenium [3]， and makes it unable to give full play to the advantages of selenium resources in the soil. It is of important significance for the optimal utilization of selenium resources in soil to study how to improve soil available selenium content.
Microorganisms play an important role in the geochemical cycle and morphological transformation of selenium [4-6]， which can metabolize selenium through a variety of ways including oxidation， reduction， assimilation and methylation [7]. It has found that a variety of microorganisms can tolerate high concentrations of selenite， such as Pseudomonas sp.， Delftia tsuruhatensis， Bacillus sphaericus， which can tolerate the selenite concentration of up to 600 mM [8]. High-selenium environment is the potential source of high-selenium-tolerant microbial population. Many strains with high-selenium tolerances have been screened from Yutangba of Enshi City， Hubei Province， China. Peng et al. [9] screened 3 strains identified as Acinetobacter baumannii， B. licheniformis and B. subtilis， which had the ability to tolerate selenium of 25 000 μg/mL. Yuan et al. [10] also screened 3 strains with the ability to reduce selenite， and found that the concentration of selenium-resistant was as high as 800 mM， which was one of the highest concentrations of tetravalent selenium that bacteria could tolerate. Studies have shown that under high selenium stress， bacteria would have stress responses， which could transform the high toxicity of tetravalent or hexavalent selenium into low-toxic elemental selenium. Therefore， the use of microorganisms to transform is possible to achieve the biological activation of soil selenium. The study on selenium-tolerant microorganisms in Enshi City， Hubei Province， started early， but there is still no report related with the microorganisms to transform soil selenium in Guangxi. Therefore， a number of soil samples were collected from selenium-riched areas in Guangxi to screen out selenium-tolerant microorganisms， with the aim to lay the foundation to obtain the natural selenium-riched microorganisms for soil selenium activation.
Materials and Methods
Test material
Strains
The tested strains were isolated and purified from the soil samples collected from a variety of selenium-riched areas， such as Yongfu， Bama， Yulin Hanshan， Guiping and Tengxian in Guangxi.
Reagents
Sodium selenite （Na2SeO3， AR 98%） was purchased from Shandong Xiya Chemical Industry Co.， Ltd. Phosphate Buffered Saline （PBS） was purchased from Shanghai Sangon Biotech Service Co.， Ltd. Other chemicals were all analytical pure products produced in China.
Selenium solution preparation： 22.35 g Na2SeO3 was taken and dissolved in deionized water and set to a volume of 100 ml. The concentration of selenium solution was 100 mg/ml. After filtered using the sterile filter （0.22 μm）， the solution was stored in the shade for later use.
Medium
The medium included bacteria， actinomycetes， fungal liquid medium and solid medium.
Bacterial liquid medium： peptone 10 g， beef extract 3 g， sodium chloride 5 g， distilled water 1 000 ml， pH （7.3 ± 0.2）. Actinomycetes liquid medium： soluble starch 20 g， sodium chloride 0.5 g， ferrous sulfate 0.01 g， potassium nitrate 1 g， dipotassium hydrogen phosphateg 0.5 g， magnesium sulfate 5 g， distilled water 1 000 ml， pH （7.3 ± 0.2）. Fungal liquid medium： 20% potato leachate 1 000 ml， sucrose 20 g. Preparation of potato leachate： 200 g peeled potato slices was taken and added with 1 000 ml of distilled water to boil 30 min. And then the juice was filtered with gauze， which was then added with the original amount of water.
Bacteria， acinomycetes and fungal solid medium： nutrient agar， Gauses synthetic media No.1， potato dextrose agar were purchased from Guangdong Huankai Microbial Sci. & Tech. Co.， Ltd..
All medium were sterilized at 121 ℃ for 20 min before use.
Test methods
Isolation of microorganisms Soil samples were collected from a number of selenium-riched areas in Yongfu， Bama， Yulin Han Shan， Guiping， Tengxian of Guangxi for microbial isolation. First， 10 g soil sample was taken and added to the 90 ml sterile PBS pre-loaded with 10 glass beads， and then oscillated at 30 ℃ of 200 r/min for 30 min. After placed still for 5 min， soil suspension was collected， which was then centrifuged at 5 000 r/min for 15 min， and then the precipitate was suspended using 10 ml PBS， which was then stored at 4 ℃ for later use. Then， 2 ml of the prepared sample was added to 100 ml of liquid medium with the selenium concentration of 100 μg/ml for screening culture. And then the gradient dilution method was used to prepare 10-2-10-6 series of diluent. And then the solid medium were coated with 100 μl of the diluent and cultured at 30 ℃ until the colonies grew. Then， a single colony was picked， and streak plate method was used to obtain strains of pure culture. Selection of selenium-tolerant strains The isolated and purified strains were inoculated to the liquid medium. And then 5 μl of 24-48 h culture solution was taken and inoculated into the solid medium with different selenium concentrations. The concentrations of selenium in the selenium-contained solid medium were set to the increase gradients of 100， 200， 300， 400， 500， 600， 700， 800， 900 and 1 000 μg/ml. After the first 10 rounds of selenium-enriched medium removal， the survived strains could be further screened at a higher concentration of selenium.
Molecular biological identification of selenium-tolerant strains
The 16S rDNA sequences of the strains were sequenced by Nanning Gold Tech Co.， Ltd. The obtained sequences were submitted to GenBank and the BLAST software was used to make similar alignments with known sequences in the GenBank library. And MEGA5. 0 was used to construct the phylogenetic tree to identify the genus of strains.
Results and Analysis
Isolation of strains
In this test， the selenium-added liquid medium was used for the initial screening of the selenium-tolerant strains， and 62 strains with different colony characteristics were isolated from a number of the collected selenium-riched soil samples by using gradient dilution plate， in which 32 strains were bacteria， 9 actinomyces， 10 molds and 11 yeasts. These strains were purified by the streak plate method， and then the purified strains were stored in the test tube slant at 4 ℃.
Selection of the tolerance to selenium
After the first 10 rounds of selenium concentration increasing screening， 38 strains were able to survive on solid media at the selenium concentration of 1 000 μg/ml， of which 26 strains were bacteria， 5 strains were molds and 7 strains were yeasts. These strains could reduce selenite to red elemental selenium and presented as red colonies on the selenium-containing plate. Among the screened strains， the actinomycetes were the worst tolerant to selenium， 2 of which only showed tolerance to the selenium concentration of 900 μg/ml at most， and when the selenium concentration reached 1 000 μg/ml， all of the actinomycetes could not grow any more. Although the 7 strains of yeasts could tolerate the selenium of 1 000 μg/ml， their growth was very weak and their colonies were dotted on the plate. On the plate with 1 000 μg/ml selenium， the growth of molds was also weakened to some extent， which was presented as the inhibition of mycelial growth. Bacteria were relatively more tolerant to selenium， and 80% of the screened bacteria could grow on the plate with a selenium concentration of 1 000 μg/ml and most of the bacteria grew well. The strains which could tolerate the 1 000 μg/ml selenium were further tested at higher selenium concentrations. The concentration of selenium on the plate was increased at the gradient of 500 μg/ml from 1 000-30 000 μg/ml. With the gradual increase of selenium concentration on the medium， the growth of strains was inhibited， and the strains could not grow when the selenium concentration was over their tolerance capacity. The results showed that the maximum tolerated selenium concentration of yeasts was 1 500 μg/ml， the mold was 3 000 μg/ml and the bacteria was 29 000 μg/ml. Among them， a total of 8 strains of bacteria resistant showed tolerance to the selenium at the concentration of over 10 000 μg/ml （Fig. 1）， of which 3 strains were tolerant to selenium concentration of 11 000 μg/ml （named YLB1-6， BMB2-1， TXB1-8， respectively）， 1 tolerant to selenium concentration of 11 500 μg/ml （GPB2-5）， 1 to selenium concentration of 15 000 μg/ml （YLB1-26）， 2 to 20 000 μg/ml （YLB1-2， YFB1-8）， and 1 to 29 000 μg/ml （YLB1-33）.
Strain identification results
Colony morphological characteristics After cultured on the nutrient agar medium at 37 ℃ for 24 h， the morphology of each strain colony was shown in Fig. 1. Strain YLB1-6 grew well， and the colonies were gray-white， round or nearly round shape with radiated melt wax margins and slightly uplifted center， opaque， of soft texture and slightly luster. Strain BMB2-1 was nearly round， faint yellow， flat with irregular margins， semi-clear with smooth and moist surface. Strain TXB1-8 was n nearly round， faint yellow， flat with irregular margins， opaque with smooth， moist and sticky surface. Strain GPB2-5 colonies were round， white， opaque with regular and smooth margins. Strains YLB1-26 and YLB1-33 were white， round， flat and opaque with jagged margins and rough and folded surface. Strains YLB1-2 and YFB1-8 were round with uplifted centers， opaque with smooth surface and regular margins， viscous， and easy to lift up.
Results of 16S DNA amplificationThe 16S rDNA sequences of the selected 8 strains of bacteria with tolerance to the selenium concentration of over 10 000 μg/ml were amplified by PCR （Fig. 2）. The 16S rDNA sequences of these strains were all about 1 450 bp in length.
Homology and phylogenetic analysis results based on 16S DNA sequence
BLAST analysis results （Table 1） showed that the similarities of the 8 strains of selenium-tolerant bacteria to their homologous strains were all more than 99%. YLB1-6 could be initially identified as B. cereus， BMB2-1 and TXB1-8 were B. pumilus， GPB2-5 was B. thuringiensis， YLB1-26 and YLB1-33 were B. licheniformis， YLB1 -2 and YFB1-8 were Serratia marcescens. The phylogenetic tree was constructed using N-J method （MEGA 5.0） based on the 16S rDNA sequences of the selenium-tolerant bacteria from the selenium-riched areas in Guangxi （Fig.3）. In the phylogenetic tree， the isolated selenium-tolerant bacteria were aggregated into two large branches， one of which was Bacillus and the other was Serratia. Among them， Bacillus was the dominant bacterial community. YLB1-6 was identified as B. cereus， BMB2-1 and TXB1-8 as B. pumilus， GPB2-5 as B. thuringiensis， YLB1-26 and YLB1-33 as B. licheniformis， and YLB1-2 and YFB1-8 as S. marcescens in combination with 16S DNA sequence analysis and phylogenetic tree analysis.
Discussion
In this study， the screening of selenium-tolerant strains in Guangxi selenium-riched area showed that the soil bacteria had the strongest selenium-tolerant capacity and actinomycetes had the worst selenium-tolerant capacity. Among the screened selenium-tolerant strains， 8 were bacteria. According to 16S rDNA sequence and phylogenetic tree analysis， 6 strains of the bacteria belonged to the commonly selenium-tolerant Bacill， and the other 2 belonged to Eubacteriae. Serratia marcescens was first reported of high selenium tolerance. The screened strains could tolerant the selenium concentration of over 10 000 μg/ml on the solid medium containing selenium， and the selenium-tolerant capacity of YLB1-33 was the best， reaching 29 000 μg/ml， which was extremely rare in selenium-riched areas of Guangxi （non-high-selenium areas）.
There are many hypotheses about the mechanism of selenium tolerance of microorganisms. Currently， a widely accepted hypothesis is that microorganisms can reduce high concentrations of selenate or selenite to low-toxic red elemental selenium through their own reduction [11-13] to reduce the toxic effects of high-selenium on the cells. The red elemental selenium is actually a water-soluble red elemental selenium-protein compound [14]， which is considered as a way of detoxification of selenium due to its low toxicity and thermal stability [15]. The red elemental selenium produced by the reduction of selenium-tolerant microorganisms has good biological activity in liver protection， immunomodulation， tumor inhibition and so on [16]， and thus it can be directly used as a health selenium supplement preparation. On the other hand， selenium-tolerant microorganisms can transform the selenium of different valence states selenium in selenium metabolism， and can transform inorganic selenium into organic selenium， which has great potential for application in soil selenium activation. Most of the selenium-riched soils in Guangxi are acidic red and reddish loam， and the soil selenium is mainly selenite， which is easily adsorbed by iron oxides and clay minerals. However， under the action of selenium-tolerant microorganisms， it is possible to transform the strong adsorbed selenite into soluble organic selenium [17]， which can release the adsorbed and fixed selenium， thereby improving the availability of soil selenium. Although the selenium-tolerant microorganisms have great potential for application in soil selenium activation， they should be further screened to obtain effective strains with high selenium transformation efficiency. Moreover， it is of important practical significance for the utilization of soil selenium resources and development of selenium-enriched agricultural products to carry out the study on the activation conditions of natural selenium-rich microorganisms and on the colonization ability of selenium-riched soil.
Conclusion
In this study， 8 strains of selenium-tolerant microorganisms were isolated and identified from the soil samples collected in a number of selenium-riched areas in Guangxi， namely， 1 strain of B. cereus， 2 strains of B. pumilus， 2 strains of B. licheniformis， 1 strain of B. thuringiensis， and 2 strains of S. marcescens， indicating that selenium-tolerant microorganisms had a population diversity. The discovery of selenium-tolerant microorganisms has potential application value for the utilization of soil resources and the development of selenium-enriched agricultural products.
References
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[2] LIU YX， YANG JH， SHI ML， et al. Analysis on the development prospects of Se-rich functional agricultural products in Guangxi[J]. Agriculture and Technology， 2015，35 （1）： 176-178.
[3] GENG JM. Chemical characteristics of selenium in paddy soils of genotypic differences and mechanism of selenium absorption and accumulation of rice in Hainan Province[D]. Hainan： Hainan University， 2010.
[4] STOLZ J， BASU P， OREMLAND R. Microbial transformation of elements： the case of arsenic and selenium[J]. International Microbiology， 2002， 5 （4）： 201-207.
[5] STOLZ J， BASU P， SANTINI JM， et al. Arsenic and selenium in microbial metabolism [J]. Annual Review of Microbiology， 2006 （60）： 107-130.
[6] XU QL， WU CL， ZHAO GS， et al. Selenium metabolism in microorganisms[J]. Microbiology， 2017， 44 （1）： 207-216.
[7] NANCHARAIAH YV， LENS PNL. Ecology and Biotechnology of Selenium-Respiring Bacteria[J]. Microbiology and Molecular Biology Re-views， 2015， 79 （1）： 61-80.
[8] GHOSH A， MOHOD AM， PAKNIKA KM， et al. Isolation and characterization of selenite-and selenate-tolerant microorganisms from selenium-contaminated sites[J]. World Journal of Microbiology & Biotechnology， 2008， 24 （8）： 1607-1611. [9] PENG QZ， FAN J， XIANG DE， et al. Isolation， screening， identification of three strains with high-se tolerance[J]. Studies of Trace Elements and Health， 2012，29 （3）： 4-6.
[10] YUAN YQ， ZHU JM， LIU CQ， et al. Three high-reducing selenite-tolerance bacteria from high-Se carbonaceous mudstone[J]. Earth Science Frontiers， 2014，21 （2）： 331-341.
[11] TOMEI FA， BARTON LL， LEMANSKI CL， et al. Transformation of selenate and selenite to elemental selenium by Desulfovibrio desulfuricans[J]. Journal of Industrial Microbiology， 1995， 14 （3/4）： 329-336.
[12] IKE M， TAKAHASH K， FUJITA T， et al. Selenate reduction by bacteria isolated from aquatic environment free from selenium contamination[J]. Water Research， 2000， 34 （11）： 3019-3025.
[13] LI BZ， LIU N， LI YQ， et al. Reduction of selenite to red elemental selenium by Rhodopseudomonas palustris strain N[J]. PLo S One， 2014，9 （4）： e95955.
[14] GAO XY， ZHANG JS， ZHANG LD. Acute toxicity and bioavailability of nano red elemental selenium[J]. Journal of Health Research， 2000， 29 （1）： 57-58.
[15] WANG DL， XIAO M， QIAN W， et al. Progress in research of the product of the red elemental selenium reduced from selenium oxy-anions by bacteria[J]. Acta Microbiologica Sinica， 2007，47 （3）： 554-557.
[16] ZHANG JS， GAO XY， ZHANG LD， et al. Study on liver protection， tumor inhibition and immune regulation of nano red elemental selenium[J]. Journal of Nutrition， 2001，23 （1）： 32-35.
[17] SHEN YC， ZHOU J. Occurrence， migration and transformation of selenium in soil[J]. Geology of Anhui， 2011，21 （3）： 186-191.
Key words Selenium-rich soil； Selenium tolerance； Strains； Identification； Guangxi
Selenium （Se） is one of the 15 trace elements essential to the human body. It is particularly important to meet the demand of peoples daily supplement of selenium by properly exploiting and utilizing the selenium resources in soil to produce natural selenium-enriched agricultural products. Guangxi is a large selenium-riched province with an area of 2.12 million hm2 of selenium-riched soil. It is the area of large contiguous selenium-riched soil currently delineated in China[1]， where the soil selenium content can reach 2.29 mg/kg at the most [2]. However， because selenium-riched soil is more widely distributed in moderately acidic soils in Guangxi， selenium is easy to form oxides and hydrous oxides with extremely low solubility with iron， which greatly reduces the available selenium [3]， and makes it unable to give full play to the advantages of selenium resources in the soil. It is of important significance for the optimal utilization of selenium resources in soil to study how to improve soil available selenium content.
Microorganisms play an important role in the geochemical cycle and morphological transformation of selenium [4-6]， which can metabolize selenium through a variety of ways including oxidation， reduction， assimilation and methylation [7]. It has found that a variety of microorganisms can tolerate high concentrations of selenite， such as Pseudomonas sp.， Delftia tsuruhatensis， Bacillus sphaericus， which can tolerate the selenite concentration of up to 600 mM [8]. High-selenium environment is the potential source of high-selenium-tolerant microbial population. Many strains with high-selenium tolerances have been screened from Yutangba of Enshi City， Hubei Province， China. Peng et al. [9] screened 3 strains identified as Acinetobacter baumannii， B. licheniformis and B. subtilis， which had the ability to tolerate selenium of 25 000 μg/mL. Yuan et al. [10] also screened 3 strains with the ability to reduce selenite， and found that the concentration of selenium-resistant was as high as 800 mM， which was one of the highest concentrations of tetravalent selenium that bacteria could tolerate. Studies have shown that under high selenium stress， bacteria would have stress responses， which could transform the high toxicity of tetravalent or hexavalent selenium into low-toxic elemental selenium. Therefore， the use of microorganisms to transform is possible to achieve the biological activation of soil selenium. The study on selenium-tolerant microorganisms in Enshi City， Hubei Province， started early， but there is still no report related with the microorganisms to transform soil selenium in Guangxi. Therefore， a number of soil samples were collected from selenium-riched areas in Guangxi to screen out selenium-tolerant microorganisms， with the aim to lay the foundation to obtain the natural selenium-riched microorganisms for soil selenium activation.
Materials and Methods
Test material
Strains
The tested strains were isolated and purified from the soil samples collected from a variety of selenium-riched areas， such as Yongfu， Bama， Yulin Hanshan， Guiping and Tengxian in Guangxi.
Reagents
Sodium selenite （Na2SeO3， AR 98%） was purchased from Shandong Xiya Chemical Industry Co.， Ltd. Phosphate Buffered Saline （PBS） was purchased from Shanghai Sangon Biotech Service Co.， Ltd. Other chemicals were all analytical pure products produced in China.
Selenium solution preparation： 22.35 g Na2SeO3 was taken and dissolved in deionized water and set to a volume of 100 ml. The concentration of selenium solution was 100 mg/ml. After filtered using the sterile filter （0.22 μm）， the solution was stored in the shade for later use.
Medium
The medium included bacteria， actinomycetes， fungal liquid medium and solid medium.
Bacterial liquid medium： peptone 10 g， beef extract 3 g， sodium chloride 5 g， distilled water 1 000 ml， pH （7.3 ± 0.2）. Actinomycetes liquid medium： soluble starch 20 g， sodium chloride 0.5 g， ferrous sulfate 0.01 g， potassium nitrate 1 g， dipotassium hydrogen phosphateg 0.5 g， magnesium sulfate 5 g， distilled water 1 000 ml， pH （7.3 ± 0.2）. Fungal liquid medium： 20% potato leachate 1 000 ml， sucrose 20 g. Preparation of potato leachate： 200 g peeled potato slices was taken and added with 1 000 ml of distilled water to boil 30 min. And then the juice was filtered with gauze， which was then added with the original amount of water.
Bacteria， acinomycetes and fungal solid medium： nutrient agar， Gauses synthetic media No.1， potato dextrose agar were purchased from Guangdong Huankai Microbial Sci. & Tech. Co.， Ltd..
All medium were sterilized at 121 ℃ for 20 min before use.
Test methods
Isolation of microorganisms Soil samples were collected from a number of selenium-riched areas in Yongfu， Bama， Yulin Han Shan， Guiping， Tengxian of Guangxi for microbial isolation. First， 10 g soil sample was taken and added to the 90 ml sterile PBS pre-loaded with 10 glass beads， and then oscillated at 30 ℃ of 200 r/min for 30 min. After placed still for 5 min， soil suspension was collected， which was then centrifuged at 5 000 r/min for 15 min， and then the precipitate was suspended using 10 ml PBS， which was then stored at 4 ℃ for later use. Then， 2 ml of the prepared sample was added to 100 ml of liquid medium with the selenium concentration of 100 μg/ml for screening culture. And then the gradient dilution method was used to prepare 10-2-10-6 series of diluent. And then the solid medium were coated with 100 μl of the diluent and cultured at 30 ℃ until the colonies grew. Then， a single colony was picked， and streak plate method was used to obtain strains of pure culture. Selection of selenium-tolerant strains The isolated and purified strains were inoculated to the liquid medium. And then 5 μl of 24-48 h culture solution was taken and inoculated into the solid medium with different selenium concentrations. The concentrations of selenium in the selenium-contained solid medium were set to the increase gradients of 100， 200， 300， 400， 500， 600， 700， 800， 900 and 1 000 μg/ml. After the first 10 rounds of selenium-enriched medium removal， the survived strains could be further screened at a higher concentration of selenium.
Molecular biological identification of selenium-tolerant strains
The 16S rDNA sequences of the strains were sequenced by Nanning Gold Tech Co.， Ltd. The obtained sequences were submitted to GenBank and the BLAST software was used to make similar alignments with known sequences in the GenBank library. And MEGA5. 0 was used to construct the phylogenetic tree to identify the genus of strains.
Results and Analysis
Isolation of strains
In this test， the selenium-added liquid medium was used for the initial screening of the selenium-tolerant strains， and 62 strains with different colony characteristics were isolated from a number of the collected selenium-riched soil samples by using gradient dilution plate， in which 32 strains were bacteria， 9 actinomyces， 10 molds and 11 yeasts. These strains were purified by the streak plate method， and then the purified strains were stored in the test tube slant at 4 ℃.
Selection of the tolerance to selenium
After the first 10 rounds of selenium concentration increasing screening， 38 strains were able to survive on solid media at the selenium concentration of 1 000 μg/ml， of which 26 strains were bacteria， 5 strains were molds and 7 strains were yeasts. These strains could reduce selenite to red elemental selenium and presented as red colonies on the selenium-containing plate. Among the screened strains， the actinomycetes were the worst tolerant to selenium， 2 of which only showed tolerance to the selenium concentration of 900 μg/ml at most， and when the selenium concentration reached 1 000 μg/ml， all of the actinomycetes could not grow any more. Although the 7 strains of yeasts could tolerate the selenium of 1 000 μg/ml， their growth was very weak and their colonies were dotted on the plate. On the plate with 1 000 μg/ml selenium， the growth of molds was also weakened to some extent， which was presented as the inhibition of mycelial growth. Bacteria were relatively more tolerant to selenium， and 80% of the screened bacteria could grow on the plate with a selenium concentration of 1 000 μg/ml and most of the bacteria grew well. The strains which could tolerate the 1 000 μg/ml selenium were further tested at higher selenium concentrations. The concentration of selenium on the plate was increased at the gradient of 500 μg/ml from 1 000-30 000 μg/ml. With the gradual increase of selenium concentration on the medium， the growth of strains was inhibited， and the strains could not grow when the selenium concentration was over their tolerance capacity. The results showed that the maximum tolerated selenium concentration of yeasts was 1 500 μg/ml， the mold was 3 000 μg/ml and the bacteria was 29 000 μg/ml. Among them， a total of 8 strains of bacteria resistant showed tolerance to the selenium at the concentration of over 10 000 μg/ml （Fig. 1）， of which 3 strains were tolerant to selenium concentration of 11 000 μg/ml （named YLB1-6， BMB2-1， TXB1-8， respectively）， 1 tolerant to selenium concentration of 11 500 μg/ml （GPB2-5）， 1 to selenium concentration of 15 000 μg/ml （YLB1-26）， 2 to 20 000 μg/ml （YLB1-2， YFB1-8）， and 1 to 29 000 μg/ml （YLB1-33）.
Strain identification results
Colony morphological characteristics After cultured on the nutrient agar medium at 37 ℃ for 24 h， the morphology of each strain colony was shown in Fig. 1. Strain YLB1-6 grew well， and the colonies were gray-white， round or nearly round shape with radiated melt wax margins and slightly uplifted center， opaque， of soft texture and slightly luster. Strain BMB2-1 was nearly round， faint yellow， flat with irregular margins， semi-clear with smooth and moist surface. Strain TXB1-8 was n nearly round， faint yellow， flat with irregular margins， opaque with smooth， moist and sticky surface. Strain GPB2-5 colonies were round， white， opaque with regular and smooth margins. Strains YLB1-26 and YLB1-33 were white， round， flat and opaque with jagged margins and rough and folded surface. Strains YLB1-2 and YFB1-8 were round with uplifted centers， opaque with smooth surface and regular margins， viscous， and easy to lift up.
Results of 16S DNA amplificationThe 16S rDNA sequences of the selected 8 strains of bacteria with tolerance to the selenium concentration of over 10 000 μg/ml were amplified by PCR （Fig. 2）. The 16S rDNA sequences of these strains were all about 1 450 bp in length.
Homology and phylogenetic analysis results based on 16S DNA sequence
BLAST analysis results （Table 1） showed that the similarities of the 8 strains of selenium-tolerant bacteria to their homologous strains were all more than 99%. YLB1-6 could be initially identified as B. cereus， BMB2-1 and TXB1-8 were B. pumilus， GPB2-5 was B. thuringiensis， YLB1-26 and YLB1-33 were B. licheniformis， YLB1 -2 and YFB1-8 were Serratia marcescens. The phylogenetic tree was constructed using N-J method （MEGA 5.0） based on the 16S rDNA sequences of the selenium-tolerant bacteria from the selenium-riched areas in Guangxi （Fig.3）. In the phylogenetic tree， the isolated selenium-tolerant bacteria were aggregated into two large branches， one of which was Bacillus and the other was Serratia. Among them， Bacillus was the dominant bacterial community. YLB1-6 was identified as B. cereus， BMB2-1 and TXB1-8 as B. pumilus， GPB2-5 as B. thuringiensis， YLB1-26 and YLB1-33 as B. licheniformis， and YLB1-2 and YFB1-8 as S. marcescens in combination with 16S DNA sequence analysis and phylogenetic tree analysis.
Discussion
In this study， the screening of selenium-tolerant strains in Guangxi selenium-riched area showed that the soil bacteria had the strongest selenium-tolerant capacity and actinomycetes had the worst selenium-tolerant capacity. Among the screened selenium-tolerant strains， 8 were bacteria. According to 16S rDNA sequence and phylogenetic tree analysis， 6 strains of the bacteria belonged to the commonly selenium-tolerant Bacill， and the other 2 belonged to Eubacteriae. Serratia marcescens was first reported of high selenium tolerance. The screened strains could tolerant the selenium concentration of over 10 000 μg/ml on the solid medium containing selenium， and the selenium-tolerant capacity of YLB1-33 was the best， reaching 29 000 μg/ml， which was extremely rare in selenium-riched areas of Guangxi （non-high-selenium areas）.
There are many hypotheses about the mechanism of selenium tolerance of microorganisms. Currently， a widely accepted hypothesis is that microorganisms can reduce high concentrations of selenate or selenite to low-toxic red elemental selenium through their own reduction [11-13] to reduce the toxic effects of high-selenium on the cells. The red elemental selenium is actually a water-soluble red elemental selenium-protein compound [14]， which is considered as a way of detoxification of selenium due to its low toxicity and thermal stability [15]. The red elemental selenium produced by the reduction of selenium-tolerant microorganisms has good biological activity in liver protection， immunomodulation， tumor inhibition and so on [16]， and thus it can be directly used as a health selenium supplement preparation. On the other hand， selenium-tolerant microorganisms can transform the selenium of different valence states selenium in selenium metabolism， and can transform inorganic selenium into organic selenium， which has great potential for application in soil selenium activation. Most of the selenium-riched soils in Guangxi are acidic red and reddish loam， and the soil selenium is mainly selenite， which is easily adsorbed by iron oxides and clay minerals. However， under the action of selenium-tolerant microorganisms， it is possible to transform the strong adsorbed selenite into soluble organic selenium [17]， which can release the adsorbed and fixed selenium， thereby improving the availability of soil selenium. Although the selenium-tolerant microorganisms have great potential for application in soil selenium activation， they should be further screened to obtain effective strains with high selenium transformation efficiency. Moreover， it is of important practical significance for the utilization of soil selenium resources and development of selenium-enriched agricultural products to carry out the study on the activation conditions of natural selenium-rich microorganisms and on the colonization ability of selenium-riched soil.
Conclusion
In this study， 8 strains of selenium-tolerant microorganisms were isolated and identified from the soil samples collected in a number of selenium-riched areas in Guangxi， namely， 1 strain of B. cereus， 2 strains of B. pumilus， 2 strains of B. licheniformis， 1 strain of B. thuringiensis， and 2 strains of S. marcescens， indicating that selenium-tolerant microorganisms had a population diversity. The discovery of selenium-tolerant microorganisms has potential application value for the utilization of soil resources and the development of selenium-enriched agricultural products.
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